BoNTs are classified as one of the six highest-risk threat agents for bioterrorism (the 'Class A agents'). BoNTs have been produced and weaponized by rogue nations and deployed by terrorist groups. As a result, specific pharmaceuticals are needed for prevention and treatment of intoxication. The goal of this application is to generate a novel Ab that can be used to prevent and treat disease caused by BoNT/A. This application builds on work showing that BoNT/A can be potently neutralized in vivo by combining three mAbs, which recognize the toxin domain (Hc), which binds cellular receptors (oligoclonal Ab). Toxin neutralization by oligoclonal Ab results from a large increase in the affinity of Ab for toxin as well as progressive blockade of the toxin surface interacting with cellular receptors. The precise contribution of these two mechanisms to toxin neutralization is unknown, as is the requirement for mAbs to the toxin-binding domain vs. mAbs to other parts of the toxin. Two important questions remain prior to producing a BoNT/A Ab therapeutic: I) Can the potency of oligoclonal Ab be reproduced in a single mAb (or mAb pair) by significantly increasing the affinity of the mAbs?; and 2) can a similar potency be achieved using mAbs to non-binding domains of the toxin? Reducing the number of mAbs would greatly simplify the complexity and cost of Ab manufacturing. Using non-binding domain mAbs would demonstrate that potent toxin neutralization does not require mAb binding toxin epitopes that interact with cellular receptors. This would simplify identification of neutralizing Abs. In addition, such mAbs would allow neutralization of second generation BoNTs, where the binding domain has been replaced with a receptor ligand. To determine the impact of affinity on BoNT/A neutralization, the affinity of two mAbs, which bind BoNT/A and neutralize toxin in vitro, will be increased at least 100 fold using in vitro mutagenesis and selection. The impact of affinity on in vitro and in vivo toxin neutralization will be determined for the single mAbs, a combination of the mAb pairs, and oligoclonal Abs. To determine the ability of non-binding domain BoNT/A Abs to neutralize toxin, phage Abs recognizing non-binding domain portions of BoNT/A will be generated and characterized with respect to affinity, epitope, and ability to neutralize toxin in vitro and in vivo. In vitro and in vivo characterization will be performed on individual mAbs, as well as combinations of binding domain and non-binding domain mAbs. Answering the above questions will generate a pharmaceutical for prevention and treatment of BoNT/A disease and also provide a route to similar pharmaceuticals for other BoNT serotypes. In addition, this approach would be applicable to four of the other Class A agents (anthrax, smallpox, plague, and hemorrhagic fever viruses).